Method of measuring a time-varying signal emission

ABSTRACT

A method of measuring a time-varying signal emission, the method including subjecting the contents of a receptacle to a thermal cycling process. During the thermal cycling process, measuring a signal emission from the contents of the receptacle at regular time intervals and recording the measured signal emission and a time stamp at each time interval. Also during the thermal cycling process, determining a temperature of the thermal cycling process at regular time intervals and recording the determined temperature and a time stamp at each time interval. The measured signal emissions are synchronized with a specific temperature of the thermal cycling process by comparing the time stamps of the measured emission signals with the time stamps of the determined temperatures.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/287,358, filed Oct. 6, 2016, now U.S. Pat. No. 10,120,136, which is adivisional of U.S. application Ser. No. 14/200,460, filed Mar. 7, 2014,now U.S. Pat. No. 9,465,161, which claims the benefit U.S. ProvisionalApplication No. 61/782,340 filed Mar. 14, 2013, the contents of each ofwhich applications is hereby incorporated by reference herein.

FIELD

This disclosure relates to an apparatus for detecting a signal emittedby each of a plurality of potential signal emission sources by indexingone or more signal detectors with respect to the signal emission sourcesto sequentially detect a signal from each signal emission source. Thedisclosure further relates to an apparatus for transmitting a signalemission from each of a plurality potential signal emission sourcesbetween first and second ends of signal transmission conduits, whereinthe first ends of the signal transmission conduits are disposed in afirst spatial arrangement, and the second ends of the signaltransmission conduits are disposed in a second spatial arrangementdifferent from the first spatial arrangement.

BACKGROUND

None of the references described or referred to herein are admitted tobe prior art to the claimed invention.

Diagnostic assays are widely used in clinical diagnosis and healthscience research to detect or quantify the presence or amount ofbiological antigens, cell or genetic abnormalities, disease states, anddisease-associated pathogens or genetic mutations in an organism orbiological sample. Where a diagnostic assay permits quantification,practitioners may be better able to calculate the extent of infection ordisease and to determine the state of a disease over time. Diagnosticassays are frequently focused on the detection of chemicals, proteins orpolysaccharides antigens, antibodies, nucleic acids, amino acids,biopolymers, cells, or tissue of interest. A variety of assays may beemployed to detect these diagnostic indicators.

Nucleic acid-based assays, in particular, generally include multiplesteps leading to the detection or quantification of one or more targetnucleic acid sequences in a sample. The targeted nucleic acid sequencesare often specific to an identifiable group of proteins, cells, tissues,organisms, or viruses, where the group is defined by at least one sharedsequence of nucleic acid that is common to members of the group and isspecific to that group in the sample being assayed. A variety of nucleicacid-based detection methods are fully described by Kohne, U.S. Pat. No.4,851,330, and Hogan, U.S. Pat. No. 5,541,308.

Detection of a targeted nucleic acid sequence frequently requires theuse of a probe comprising a nucleic acid molecule having a nucleotidebase sequence that is substantially complementary to at least a portionof the targeted sequence or its complement. Under selective assayconditions, the probe will hybridize to the targeted sequence or itscomplement in a manner permitting a practitioner to detect the presenceof the targeted sequence in a sample. Techniques of effective probepreparation are known in the art. In general, however, effective probesare designed to prevent non-specific hybridization with itself or anynucleic acid molecule that will interfere with detecting the presence ofthe targeted sequence. Probes may include, for example, a label capableof detection, where the label is, for example, a radiolabel, afluorophore or fluorescent dye, biotin, an enzyme, a chemiluminescentcompound, or another type of detectable signal known in the art.

To detect different nucleic acids of interest in a single assay,different probes configured to hybridize to different nucleic acids,each of which may provide detectibly different signals can be used. Forexample, different probes configured to hybridize to different targetscan be formulated with fluorophores that fluoresce at a predeterminedwavelength when exposed to excitation light of a prescribed excitationwavelength. Assays for detecting different target nucleic acids can beperformed in parallel by alternately exposing the sample material todifferent excitation wavelengths and detecting the level of fluorescenceat the wavelength of interest corresponding to the probe for each targetnucleic acid during the real-time monitoring process. Parallelprocessing can be performed using different signal detecting devicesconstructed and arranged to periodically measure signal emissions duringthe amplification process, and with different signal detecting devicesbeing configured to generate excitation signals of different wavelengthsand to measure emission signals of different wavelengths.

Because the probe hybridizes to the targeted sequence or its complementin a manner permitting detection of a signal indicating the presence ofthe targeted sequence in a sample, the strength of the signal isproportional to the amount of target sequence or its complement that ispresent. Accordingly, by periodically measuring, during an amplificationprocess, a signal indicative of the presence of amplicon, the growth ofamplicon overtime can be detected. Based on the data collected duringthis “real-time” monitoring of the amplification process, the amount ofthe target nucleic acid that was originally in the sample can beascertained. Exemplary systems and methods for real time detection andfor processing real time data to ascertain nucleic acid levels aredescribed, for example, in Lair, et al., U.S. Pat. No. 7,932,081,“Signal measuring system for conducting real-time amplificationassays.”.

Challenges may arise, however, when measuring emission signals during anamplification process or other process. The target sequence or itscomplement, or other emission signal source, may be contained in areceptacle that is held within an incubator or other processing modulethat is fully or partially enclosed and for which access by a signaldetector to the receptacle or other source for measuring the emissionsignal may not be practical. Moreover, for space utilizationefficiencies and/or other efficiencies (such as thermal efficiencies),the receptacles or other emission signal sources may positioned in aspatial arrangement for which it is not efficient or practical to placea signal detector in operative position to measure the emission signals.For example, a plurality of receptacles or emission signal sources maybe arranged in a rectangular arrangement whereby the receptacles areclosely spaced in multiple rows of two or more receptacles each. In sucha spatial arrangement, it may not be practical or efficient to provide asignal detector for each receptacle position or to move a signaldetector with respect to the receptacle positions to sequentiallymeasure signal emissions from each of the receptacles.

SUMMARY

Aspects of the disclosure are embodied in an apparatus for detecting asignal emission from each of a plurality of potential signal emissionsources. The apparatus comprises a plurality of signal transmissionconduits, a conduit reformatter, one or more signal detectors, and asignal detector carrier. The signal transmission conduits correspond innumber to the number of signal emission sources. Each signaltransmission conduit is associated with at least one of the signalemission sources and is configured to transmit a signal emitted by theassociated signal emission source between a first end and a second endthereof. The conduit reformatter is constructed and arranged to securethe first ends of the respective signal transmission conduits in a firstspatial arrangement corresponding to a spatial arrangement of the signalemission sources, such that the first end of each signal transmissionconduit is positioned to receive an emission signal emitted by anassociated signal emission source, and to secure the second ends of therespective signal transmission conduits in a second spatial arrangementdifferent from the first spatial arrangement. The signal detectors areconfigured to detect a signal emitted by each signal emission source.The signal detector carrier is configured to carry at least a portion ofthe one or more signal detectors and to move at least a portion of eachsignal detector in a path that sequentially places the signal detectorin signal detecting positions with respect to the second ends of thesignal transmission conduits arranged in the second spatial arrangement.

According to further aspects of the disclosure, the signal emission isan optical signal and the signal transmission conduits comprise opticalfibers.

According to further aspects of the disclosure, the first spatialarrangement is rectangular and comprises two or more rows, each rowincluding two or more of the first ends of the signal transmissionconduits.

According to further aspects of the disclosure, the second spatialarrangement comprises one or more circles, whereby the second ends of aplurality of signal transmission conduits are positioned about thecircumference of a circle.

According to further aspects of the disclosure, the second spatialarrangement comprises one or more bundles whereby the second ends of aplurality of signal transmission conduits are collected in a bundlewherein the second ends of the transmission fibers in the bundle are inclose proximity to each other.

According to further aspects of the disclosure, the signal detectorcarrier comprises a carousel configured to move at least a portion ofthe one or more signal detectors in a path corresponding to the one ormore circles of the second spatial arrangement.

According to further aspects of the disclosure, the conduit reformattercomprises a reformatter frame comprising an interface plate configuredto secure the first ends of the respective signal transmission conduitsin the first spatial arrangement, a base configured to secure the firstends of the respective signal transmission conduits in the secondspatial arrangement, and a side structure connecting the interface plateto the base at spaced-apart positions with respect to each other.

According to further aspects of the disclosure, the apparatus furthercomprises heat dissipating fins extending from the interface plate.

According to further aspects of the disclosure, the apparatus furthercomprises a signal coupling element operatively disposed with respect tothe first end of each signal transmission conduit.

According to further aspects of the disclosure, the signal detectorcarrier is constructed and arranged to be rotatable about an axis ofrotation so as to move each of the one or more signal detectors in acircular path, and the apparatus further comprises a detector carrierdrive operatively associated with the signal detector carrier. Thedetector carrier drive comprises a motor, a drive pulley coupled to orpart of the signal detector carrier such that rotation of the drivepulley causes a corresponding rotation of the signal detector carrier,and a belt operatively coupling the motor to the drive pulley.

According to further aspects of the disclosure, the detector carrierdrive further comprises a home position detector configured to detect arotational position of the detector carrier.

According to further aspects of the disclosure, the signal detectorcarrier is configured to rotate about an axis of rotation, and theapparatus further comprises a rotary connector transmitting power and/ordata between the one more signal detectors carried on the signaldetector carrier and a non-rotating data processor and/or power source.

According to further aspects of the disclosure, the rotary connectorcomprises a slip ring connector.

According to further aspects of the disclosure, the each signal emissionsource comprises a substance that emits light of a predeterminedemission wavelength when subjected to an excitation light of apredetermined excitation wavelength, and the signal detector isconfigured to generate an excitation light of the predeterminedexcitation wavelength and detect light of the predetermined emissionwavelength.

According to further aspects of the disclosure, the apparatus comprisesmore than one signal detector, each configured to generate an excitationlight of a different predetermined excitation wavelength and to detectlight of a different predetermined emission wavelength.

According to further aspects of the disclosure, each of the signalemission sources is in optical communication with a single signaltransmission conduit.

According to further aspects of the disclosure, each of the plurality ofsignal transmission conduits transmits both an excitation and anemission signal.

According to further aspects of the disclosure, the each signal detectorcomprises an excitation source carried on the signal detector carrierand configured to generate an excitation signal, excitation opticscomponents carried on the signal detector carrier and configured todirect an excitation signal from the excitation source to the second endof a signal transmission conduit when the signal detector is in a signaldetecting position with respect to the second end of the transmissionconduit, emission optics components carried on the signal detectorcarrier and configured to direct an emission signal transmitted by asignal transmission conduit when the signal detector is in a signaldetecting position with respect to the second end of the transmissionconduit, and an emission detector configured to detect an emissionsignal directed by the emission optics components from the second end ofthe transmission conduit to the emission detector when the signaldetector is in a signal detecting position with respect to the secondend of the transmission conduit.

According to further aspects of the disclosure, the emission detector iscarried on the signal detector carrier.

According to further aspects of the disclosure, the emission detectorcomprises a photodiode.

According to further aspects of the disclosure, the emission detector isfixed and disposed adjacent to the signal detector carrier.

According to further aspects of the disclosure, the emission detectorcomprises a camera.

According to further aspects of the disclosure, the emission detector isassociated with at least one excitation source and is configured todetect an emission signal transmitted by a single transmission conduit.

According to further aspects of the disclosure, the signal detectorcarrier is configured to selectively place each set of excitation opticscomponents into operative association with the emission detector, andthe emission detector is configured to detect an emission signaltransmitted by all single transmission conduits simultaneously.

Further aspects of the disclosure are embodied in an apparatus fortransmitting a signal emission from each of a plurality of potentialsignal emission sources. The apparatus comprises a plurality of signaltransmission conduits and a conduit reformatter. Each signaltransmission conduit is configured to transmit a signal emitted by oneor more of the signal emission sources between a first end and a secondend thereof. The conduit reformatter is constructed and arranged tosecure the first ends of the respective signal transmission conduits ina first spatial arrangement corresponding to a spatial arrangement ofthe signal emission sources, such that the first end of each signaltransmission conduit is positioned to receive an emission signal emittedby one or more of the signal emission sources, and to secure the secondends of the respective signal transmission conduits in a second spatialarrangement different from the first spatial arrangement.

According to further aspects of the disclosure, the signal emission isan optical signal and the signal transmission conduits comprise opticalfibers.

According to further aspects of the disclosure, the first spatialarrangement is rectangular and comprises two or more rows, each rowincluding two or more of the first ends of the signal transmissionconduits.

According to further aspects of the disclosure, the second spatialarrangement comprises one or more circles, whereby the second ends of aplurality of signal transmission conduits are positioned about thecircumference of a circle.

According to further aspects of the disclosure, the second spatialarrangement comprises one or more bundles whereby the second ends of aplurality of signal transmission conduits are collected in a bundlewherein the second ends of the transmission fibers in the bundle are inclose proximity to each other.

According to further aspects of the disclosure, the conduit reformattercomprises a reformatter frame comprising an interface plate configuredto secure the first ends of the respective signal transmission conduitsin the first spatial arrangement, a base configured to secure the firstends of the respective signal transmission conduits in the secondspatial arrangement, and a side structure connecting the interface plateto the base at spaced-apart positions with respect to each other.

According to further aspects of the disclosure, the apparatus furthercomprises heat dissipating fins extending from the interface plate.

According to further aspects of the disclosure, the apparatus furthercomprises a signal coupling element operatively disposed with respect tothe first end of each signal transmission conduit.

Further aspects of the disclosure are embodied in a method of measuringat least one time-varying signal emission from the contents of areceptacle while the contents are subject to repeated cycles oftemperature variations. The method comprises measuring the signalemission from the contents of the receptacle at repeating intervals oftime and recording the signal emission measurement and a time stamp ateach interval, recording the temperature to which the contents of thereceptacle are subjected at repeating intervals of time and recordingthe time stamp at each interval, and synchronizing the signal emissionto a specific temperature by comparing the time stamps of the signalemission measurements to time stamps of the recorded temperaturecorresponding to the specific temperature.

Further aspects of the disclosure are embodied in an apparatus fordetecting an emission signal from each of a plurality of emission signalsources, wherein each emission signal is excited by an excitationsignal. The apparatus comprises one or more excitation sourcesconfigured to generate an excitation signal that is directed at anemission signal source, one or more emission detectors, each emissiondetector being associated with at least one excitation source and beingconfigured to detect an emission signal emitted by an excitation sourceand excited by the excitation signal generated by the associatedexcitation signal source, and a carrier configured to move the one ormore excitation sources and the one or more emission detectors relativeto the emission signal sources to thereby index each emission detectorand associated excitation source past each of the emission signalsources.

According to further aspects of the disclosure, the each emission signalsource comprises a substance that emits light of a predeterminedemission wavelength when subjected to an excitation signal of apredetermined excitation wavelength and each excitation source isconfigured to generate an excitation light of the predeterminedexcitation wavelength and each associated emission detector isconfigured to detect light of the predetermined emission wavelength.

According to further aspects of the disclosure, the apparatus comprisesmore than one excitation source, each configured to generate anexcitation light of a different predetermined excitation wavelength, andmore than one associated emission detector, each configured to detectlight of a different predetermined emission wavelength.

According to further aspects of the disclosure, the carrier isconfigured to rotate about an axis of rotation and move each emissiondetector and associated excitation source in a circular path.

Other features and characteristics of the present disclosure, as well asthe methods of operation, functions of related elements of structure andthe combination of parts, and economies of manufacture, will become moreapparent upon consideration of the following description and theappended claims with reference to the accompanying drawings, all ofwhich form a part of this specification, wherein like reference numeralsdesignate corresponding parts in the various figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various, non-limiting embodiments ofthe present disclosure. In the drawings, common reference numbersindicate identical or functionally similar elements.

FIG. 1 is a perspective view of a signal detection module embodyingaspects of the present disclosure.

FIG. 2 is a front perspective view of a signal detection moduleembodying aspects of the present disclosure and according to analternate embodiment.

FIG. 3 is a rear perspective view of the signal detection module shownin FIG. 2.

FIG. 4 is a transverse cross-section of the signal detection modulealong the line IV-IV in FIG. 2.

FIG. 5 is a front perspective view of a fiber reformatter and interfaceplate of the signal detection module shown in FIGS. 2-4.

FIG. 6 is a rear perspective view of a fiber reformatter and interfaceplate shown in FIG. 5.

FIG. 7 is a top perspective view of an alternate embodiment of a fiberreformatter.

FIG. 8 shows the fiber position mapping in the interface plate of thefiber reformatter shown in FIG. 7.

FIG. 9 shows the fiber position mapping in the baseplate of the fiberreformatter shown in FIG. 7.

FIG. 10 is a table showing mapping between the interface fiber positionsand the baseplate fiber positions shown in FIGS. 8 and 9.

FIG. 11 is a top perspective of an alternate embodiment of a fiberreformatter.

FIG. 12 is a perspective view of a signal detector head.

FIG. 13 is a transverse cross-section of the signal detector head alongthe line XIII-XIII in FIG. 12.

FIG. 14 is a schematic view of an embodiment of an exemplary opticalpath within a signal detector.

FIG. 15 is a schematic view of the signal detection module embodyingaspects of the present disclosure and a power and data control systemincorporated therewith.

FIG. 16 is a schematic view of a control system for the signal detectorhead.

FIG. 17 is a perspective view of an alternate embodiment of a signaldetector head.

FIG. 18 is cross sectional view of the signal detector head of FIG. 17.

DETAILED DESCRIPTION

Unless defined otherwise, all terms of art, notations and otherscientific terms or terminology used herein have the same meaning as iscommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. Many of the techniques and procedures described orreferenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. All patents,applications, published applications and other publications referred toherein are incorporated by reference in their entirety. If a definitionset forth in this section is contrary to or otherwise inconsistent witha definition set forth in the patents, applications, publishedapplications, and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

This description may use relative spatial and/or orientation terms indescribing the position and/or orientation of a component, apparatus,location, feature, or a portion thereof. Unless specifically stated, orotherwise dictated by the context of the description, such terms,including, without limitation, top, bottom, above, below, under, on topof, upper, lower, left of, right of, in front of, behind, next to,adjacent, between, horizontal, vertical, diagonal, longitudinal,transverse, etc., are used for convenience in referring to suchcomponent, apparatus, location, feature, or a portion thereof in thedrawings and are not intended to be limiting.

Nucleic Acid Diagnostic Assays

Aspects of the present disclosure involve apparatus and procedures fortransmitting and/or measuring signals emitted by potential emissionsignal sources and can be used in conjunction with nucleic aciddiagnostic assays, including “real-time” amplification assays and“end-point” amplification assays.

There are many established procedures in use for amplifying nucleicacids, including the polymerase chain reaction (PCR), (see, e.g.,Mullis, “Process for Amplifying, Detecting, and/or Cloning Nucleic AcidSequences,” U.S. Pat. No. 4,683,195), transcription-mediatedamplification (TMA), (see, e.g., Kacian et al., “Nucleic Acid SequenceAmplification Methods,” U.S. Pat. No. 5,399,491), ligase chain reaction(LCR), (see, e.g., Birkenmeyer, “Amplification of Target Nucleic AcidsUsing Gap Filling Ligase Chain Reaction,” U.S. Pat. No. 5,427,930),strand displacement amplification (SDA), (see, e.g., Walker, “StrandDisplacement Amplification,” U.S. Pat. No. 5,455,166), and loop-mediatedisothermal amplification (see, e.g., Notomi et al., “Process forSynthesizing Nucleic Acid,” U.S. Pat. No. 6,410,278). A review ofseveral amplification procedures currently in use, including PCR andTMA, is provided in HELEN H. LEE ET AL., NUCLEIC ACID AMPLIFICATIONTECHNOLOGIES (1997).

Real-time amplification assays can be used to determine the presence andamount of a target nucleic acid in a sample which, by way of example, isderived from a pathogenic organism or virus. By determining the quantityof a target nucleic acid in a sample, a practitioner can approximate theamount or load of the organism or virus in the sample. In oneapplication, a real-time amplification assay may be used to screen bloodor blood products intended for transfusion for bloodborne pathogens,such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV).In another application, a real-time assay may be used to monitor theefficacy of a therapeutic regimen in a patient infected with apathogenic organism or virus, or that is afflicted with a diseasecharacterized by aberrant or mutant gene expression. Real-timeamplification assays may also be used for diagnostic purposes, as wellas in gene expression determinations. Exemplary systems and methods forperforming real-time amplification assays are described in U.S. Pat. No.7,897,337, entitled “Methods for Performing Multi-Formatted Assays,” andin U.S. Pat. No. 8,008,066, entitled, “System for performingmulti-formatted assays.”

In addition to implementation of embodiments of the disclosure inconjunction with real-time amplification assays, embodiments of thedisclosure may also be implemented in conjunction with end pointamplification assays. In end-point amplification assays, the presence ofamplification products containing the target sequence or its complementis determined at the conclusion of an amplification procedure. Exemplarysystems and methods for end-point detection are described in U.S. Pat.No. 6,335,166, entitled “Automated Process For Isolating and Amplifyinga Target Nucleic Acid Sequence.” In contrast, in “real-time”amplification assays, the amount of amplification products containingthe target sequence or its complement is determined during anamplification procedure. In the real-time amplification assay, theconcentration of a target nucleic acid can be determined using dataacquired by making periodic measurements of signals that are functionsof the amount of amplification product in the sample containing thetarget sequence, or its complement, and calculating the rate at whichthe target sequence is being amplified from the acquired data.

For real-time amplification assays, the probes are, in certainembodiments, unimolecular, self-hybridizing probes having a pair ofinteracting labels that interact and thereby emit different signals,depending on whether the probes are in a self-hybridized state orhybridized to the target sequence or its complement. See, e.g., Diamondet al., “Displacement Polynucleotide Assay Method and PolynucleotideComplex Reagent Therefor,” U.S. Pat. No. 4,766,062; Tyagi et al.,“Detectably Labeled Dual Conformation Oligonucleotide Probes, Assays andKits,” U.S. Pat. No. 5,925,517; Tyagi et al., “Nucleic Acid DetectionProbes Having Non-FRET Fluorescence Quenching and Kits and AssaysIncluding Such Probes,” U.S. Pat. No. 6,150,097; and Becker et al.,“Molecular Torches,” U.S. Pat. No. 6,361,945. Other probes are known,including complementary, bimolecular probes, probes labeled with anintercalating dye and the use of intercalating dyes to distinguishbetween single-stranded and double-stranded nucleic acids. See, e.g.,Morrison, “Competitive Homogenous Assay,” U.S. Pat. No. 5,928,862;Higuchi, “Homogenous Methods for Nucleic Acid Amplification andDetection,” U.S. Pat. No. 5,994,056; and Yokoyama et al., “Method forAssaying Nucleic Acid,” U.S. Pat. No. 6,541,205. Examples of interactinglabels include enzyme/substrate, enzyme/cofactor, luminescent/quencher,luminescent/adduct, dye dimers and Forrester energy transfer pairs.Methods and materials for joining interacting labels to probes foroptimal signal differentiation are described in the above-citedreferences. A variety of different labeled probes and probing mechanismsare known in the art, including those where the probe does not hybridizeto the target sequence. See, e.g., U.S. Pat. No. 5,846,717 and PCTPublication No. 2012096523. The embodiments of the present disclosureoperate regardless of the particular labeling scheme utilized providedthe moiety to be detected can be excited by a particular wavelength oflight and emits a distinguishable emission spectra.

In an exemplary real-time amplification assay, the interacting labelsinclude a fluorescent moiety, or other emission moiety, and a quenchermoiety, such as, for example, 4-(4-dimethylaminophenylazo) benzoic acid(DABCYL). The fluorescent moiety emits light energy (i.e., fluoresces)at a specific emission wavelength when excited by light energy at anappropriate excitation wavelength. When the fluorescent moiety and thequencher moiety are held in close proximity, light energy emitted by thefluorescent moiety is absorbed by the quencher moiety. But when a probehybridizes to a nucleic acid present in the sample, the fluorescent andquencher moieties are separated from each other and light energy emittedby the fluorescent moiety can be detected. Fluorescent moieties havingdifferent and distinguishable excitation and emission wavelengths areoften combined with different probes. The different probes can be addedto a sample, and the presence and amount of target nucleic acidsassociated with each probe can be determined by alternately exposing thesample to light energy at different excitation wavelengths and measuringthe light emission from the sample at the different wavelengthscorresponding to the different fluorescent moieties. In anotherembodiment, different fluorescent moieties having the same excitationwavelength, but different and distinguishable emission wavelengths arecombined with different probes. The presence and amount of targetnucleic acids associated with each probe can be determined by exposingthe sample to a specific wavelength light energy and the light emissionfrom the sample at the different wavelengths corresponding to thedifferent fluorescent moieties is measured.

In one example of a multiplex, real-time amplification assay, thefollowing may be added to a sample prior to initiating the amplificationreaction: a first probe having a quencher moiety and a first fluorescentdye (having an excitation wavelength λ_(ex1) and emission wavelengthλ_(em1)) joined to its 5′ and 3′ ends and having specificity for anucleic acid sequence derived from HCV; a second probe having a quenchermoiety and a second fluorescent dye (having an excitation wavelengthλ_(ex2) and emission wavelength λ_(em2)) joined to its 5′ and 3′ endsand having specificity for a nucleic acid sequence derived from HIV Type1 (HIV-1); and a third probe having a quencher moiety and a thirdfluorescent dye (having an excitation wavelength λ_(ex3) and emissionwavelength λ_(em3)) joined to its 5′ and 3′ ends and having specificityfor a nucleic acid sequence derived from West Nile virus (WNV). Aftercombining the probes in a sample with amplification reagents, thesamples can be periodically and alternately exposed to excitation lightat wavelengths λ_(ex1), λ_(ex2), and λ_(ex3), and then measured foremission light at wavelengths λ_(em1), λ_(em2), and λ_(em3), to detectthe presence (or absence) and amount of all three viruses in the singlesample. The components of an amplification reagent will depend on theassay to be performed, but will generally contain at least oneamplification oligonucleotide, such as a primer, a promoter-primer,and/or a promoter oligonucleotide, nucleoside triphosphates, andcofactors, such as magnesium ions, in a suitable buffer.

Where an amplification procedure is used to increase the amount oftarget sequence, or its complement, present in a sample before detectioncan occur, it is desirable to include a “control” to ensure thatamplification has taken place. Such a control can be a known nucleicacid sequence that is unrelated to the sequence(s) of interest. A probe(i.e., a control probe) having specificity for the control sequence andhaving a unique fluorescent dye (i.e., the control dye) and quenchercombination is added to the sample, along with one or more amplificationreagents needed to amplify the control sequence, as well as the targetsequence(s). After exposing the sample to appropriate amplificationconditions, the sample is alternately exposed to light energy atdifferent excitation wavelengths (including the excitation wavelengthfor the control dye) and emission light is detected. Detection ofemission light of a wavelength corresponding to the control dye confirmsthat the amplification was successful (i.e., the control sequence wasindeed amplified), and thus, any failure to detect emission lightcorresponding to the probe(s) of the target sequence(s) is not likelydue to a failed amplification. Conversely, failure to detect emissionlight from the control dye may be indicative of a failed amplification,thus calling into question the results from that assay. Alternatively,failure to detect emission light may be due to failure or deterioratedmechanical and/or electrical performance of an instrument (describedbelow) for detecting the emission light.

Apparatus and procedures embodying aspects of the disclosure may be useda variety of nucleic acid amplification procedures, including inconjunction with real-time PCR, which requires accurate/rapidthermocycling between denaturation (˜95° C.), annealing (˜55° C.), andsynthesis (˜72° C.) temperatures. For this purpose, receptaclescontaining a reaction mixture that is to be subject to PCR are held in athermocycler configured to effect temperature cycling between thedenaturation, annealing, and synthesis phases. Emission signalmonitoring (e.g., of fluorescence) of the contents of the receptaclesheld in the thermocycler occurs at one or many color wavelengths duringeach temperature cycle between 95° C., 55° C., and synthesis 72° C. PCRcomponents include; for example, a forward and a reverse amplificationoligonucleotides, and a labeled poly or oligonucleotide probe. Duringone exemplary PCR procedure, nucleic acid amplification oligonucleotideshybridize to opposite strands of a target nucleic acid and are orientedwith their 3′ ends facing each other so that synthesis by apolymerization enzyme such as a polymerase extends across the segment ofnucleic acid between them. While the probe is intact, the proximity ofthe quencher dye quenches the fluorescence of the reporter dye. Duringamplification if the target sequence is present, the fluorogenic probeanneals downstream from one of the amplification oligonucleotide sitesand is cleaved by the 5′ nuclease activity of the polymerization enzymeduring amplification oligonucleotide extension. The cleavage of theprobe separates the reporter dye from the quencher dye, thus renderingdetectable the reporter dye signal and, eventually, removing the probefrom the target strand, allowing amplification oligonucleotide extensionto continue to the end of the template strand.

One round of PCR synthesis will result in new strands of indeterminatelength which, like the parental strands, can hybridize to theamplification oligonucleotides upon denaturation and annealing. Theseproducts accumulate arithmetically with each subsequence cycle ofdenaturation, annealing to amplification oligonucleotides, andsynthesis. The second cycle of denaturation, annealing, and synthesisproduces two single-stranded products that together compose a discretedouble-stranded product which comprises the length between theamplification oligonucleotide ends. Each strand of this discrete productis complementary to one of the two amplification oligonucleotides andcan therefore participate as a template in subsequent cycles. The amountof this product doubles with every subsequent cycle of synthesis,denaturation and annealing. This accumulates exponentially so that 30cycles should result in a 2²⁸-fold (270 million-fold) amplification ofthe discrete product.

Signal Detection Module/Fiber Reformatter

Detection, and, optionally, measurement, of emission signals fromemission signal sources, such as receptacles containing reactionmaterials undergoing amplification as described above can be performedin accordance with aspects of the present disclosure with a signaldetection module. A signal detection module embodying aspects of thepresent disclosure is indicated by reference number 100 in FIG. 1. Thesignal detection module includes an upright reformatter frame 150. Twosignal detector heads 200 are attached to a lower end of the reformatterframe 150 and an interface plate 160 is attached to an upper end of thereformatter frame 150. In general, the reformatter frame includes sides152, 154 which, in the illustrated embodiment, comprise generallyvertical columns, and a base 156 within which are formed a plurality offiber-positioning holes 158. Note that the designation of thereformatter frame 150 as being upright or the sides 152, 154 as beingvertical is merely to provide a convenient reference with respect to theorientation of the signal detection module 100 as shown in FIG. 1, andsuch terms of orientation are not intended to be limiting. Accordingly,the signal detection module 100 could be oriented at any angle,including vertical or horizontal, or any angle therebetween. Thereformatter frame has a variety of purposes, including organizing andarranging a plurality of optical transmission fibers 180 between anexcitation/emission area and a detection area in an optimum opticalpathway orientation. In particular embodiments the reformatter alsoprovides for controlled orientation of a plurality of opticaltransmission fibers 180 between the fins of a heat sink to a detectionarea.

Signal transmission conduits, such as optical transmission fibers 180extend between the interface plate 160 and the base 156 of thereformatter frame 150. In the present context, an optical transmissionfiber, or optical fiber, comprises a flexible, transparent rod made ofglass (silica) or plastic that functions as a waveguide, or light pipe,to transmit light between the two ends of the fiber. Optical fiberstypically include a transparent core surrounded by an opaque ortransparent cladding material having a lower index of refraction thanthe core material. A light transmission is maintained within the core bytotal internal reflection. Each optical fiber may comprise a singlefiber having a single fiber core, or each fiber may comprise a fiberbundle of two or more fibers. Fiber bundlers may be preferred if a tightbend radius is required for the transmission fiber 180. In certainembodiments it may be preferable to provide an optical fiber claddingthat is resistant to the effects of high heat indexes in that theoptical transmission properties of the fiber are maintained in thepresence of heat indexes well-above room temperature.

In one aspect of the disclosure, the reformatter frame is constructedand arranged to reconfigure the relative spatial arrangements of thefibers 180 from their first ends to their second ends so as to rearrangethe transmission fibers 180 into a spatial arrangement in which they canbe more efficiently interrogated by a signal measuring device to measurea signal transmitted therethrough. In the context of this description,the first end of the fiber 180 corresponds to the end of the fiberclosest to the signal emission source is being measured, and the secondend of the fiber corresponds to the end of the fiber closest to thesignal detector. This is merely a convenient terminology fordistinguishing one end of the transmission fiber 180 from another end ofthe transmission fiber 180. Otherwise, the designation of the ends ofthe fibers as being a first end or a second end is arbitrary.

The first ends of the transmission fibers 180 are attached to theinterface plate 160, for example extending into or through openingsformed through the interface plate 160. Signal coupling elements 162,e.g., ferrules, may be provided in each of the openings formed in theinterface plate 160 for securely attaching each optical transmissionfiber 180 to the interface plate 160. Although not shown in FIG. 1, eachopening formed in the interface plate 160 may be in signal transmissioncommunication with an emission signal source. In one embodiment, asignal emission source may comprise a receptacle containing the contentsof a chemical or biological assay. In the case of optical emissionsignals, the receptacles may be positioned and held so as to opticallyisolate each receptacle from the surrounding receptacles. In addition,as noted above, the receptacles may be held within an incubator devicelocated in optical communication with the interface plate 160,configured to alter the temperature of receptacles or maintain thereceptacles at a specified temperature. In such an application, it maybe desirable that the interface plate 160 is formed of a suitablyheat-conducting material, such as aluminum or copper, and that theinterface plate 160 further include heat dissipating fins 164 formed onone side of the interface plate 160 for dissipating heat from theinterface plate 160 by convection. Also, coupling elements (ferrules)162 may be thermally insulating to insulate the transmission fibers 180from the heat of the receptacles held within the incubator. Suitableinsulating materials include Ultem (polyethylene ketone (PEEK)).

In the embodiment illustrated in FIG. 1, the transmission fibers 180 areattached to the interface plate 160 in a rectangular configurationcomprising a plurality of rows, each row having one or more transmissionfibers 180. As shown in the illustrated embodiment, in an application inwhich the interface plate 160 includes heat dissipating fins 164, thetransmission fibers 180 may extend between adjacent fins 164 into anassociated opening formed in the interface plate 160. The illustratedembodiment includes twelve rows of five transmission fibers 180 each,for a total of sixty transmission fibers that can be employed forinterrogating up to sixty individual emission sources, such as reactionreceptacles containing reaction materials therein. Each row oftransmission fibers 180 may be disposed between a pair of adjacentheat-dissipating fins 164.

The second ends of the transmission fibers 180 are connected to the base156 of the reformatter frame 150, for example, by being aligned with orinserted into or through fiber-positioning holes 158. Thefiber-positioning holes 158 are in a spatial arrangement that isdifferent from the spatial arrangement fiber-receiving holes formed inthe interface plate 160 and are in a position that can be moreefficiently interrogated by one or more signal detectors. In theillustrated embodiment, each of the fiber position holes 158 is arrangedin a circle, FIG. 1 exemplifies two such arrangements, each circleaccommodating a plurality of the transmission fibers 180 extending fromthe interface plate 160. Other spatial arrangements are contemplated,including, two or more concentric circles, one or more open rectangles,one or more ovals, etc.

The length of the fiber reformatter 150 is defined by the distancebetween the base 156 and the interface plate 160 and is selected bybalancing two, sometimes competing considerations. On the one hand, tomake the signal detection module 100 as compact as possible, thesmallest possible length of the fiber reformatter 150 is desired. On theother hand, because the flexibility of the transmission fibers 180 maybe limited, a longer fiber reformatter 150 will make it easier to bendeach transmission fiber 180 when reformatting the fiber from itsposition within the fiber arrangement in the interface plate 160 to itsposition in the fiber arrangement in the base 156 of the fiberreformatter 150. In one embodiment, using thirty fibers having adiameter of 1.5 mm, a fiber reformatter having a length of 200-300 mmwas found to be suitable. In other embodiments, plastic fibers having adiameter of 1.5 mm and a length of 165 mm+/−10 mm were used.

A somewhat modified embodiment of the signal detection module embodyingaspects of the present disclosure is represented by reference number 600in FIGS. 2, 3, and 4. The signal detection module 600 includes areformatter frame 650 that includes sides 652, 654 and a base 656. Aninterface plate 660 is attached to one end of the reformatter frame 650,and two signal detector heads 200 are attached to the base 656 at anopposite end of the reformatter frame 650. As opposed to the embodimentshow in FIG. 1, in which the base 156 of the reformatter frame 150 formsa generally orthogonal angle with respect to the sides 152,154 of thereformatter frame 150 such that the base 156 is generally parallel tothe interface plate 160, the reformatter frame 650 of signal detectionmodule 600 is configured such that the base 656 is at an acute anglewith respect to the sides 652, 654 so that the base 656 is not parallelto the interface plate 660.

Transmission fibers 180 extend from a first end thereof connected to theinterface plate 660 in a first spatial arrangement to a second endthereof connected to the base 656 in a second spatial arrangement. Aswith the embodiment shown in FIG. 1, the transmission fibers 180 arereformatted from a generally rectangular configuration attached to theinterface plate 660 into two circular arrangements, each accommodatinghalf of the transmission fibers 180, attached to the base 656.

As also shown in FIGS. 2 to 4, a processing module 500, such as anincubator, including a plurality of receptacle holders 502, eachconfigured to hold one or more receptacles 504, is positioned above theinterface plate 660. In the illustrated embodiment, the receptacleholders 502 are constructed and arranged to hold sixty receptacles 504arranged in twelve rows of five receptacles 504 each. In one embodiment,processing module 500 may be an incubator, and each receptacle holder502 may be constructed and arranged to impart thermal energy to thereceptacles 504 held thereby to change and/or maintain the temperatureof the contents of each receptacle 504. In one embodiment, processingmodule 500 comprises an incubator as disclosed in application Ser. No.61/677,976, filed on Jul. 31, 2012, to the extent published in U.S.Patent Application Publication No. 2014-0038192, which claims prioritytherefrom.

For applications in which heat dissipation from the interface plate 660is necessary or desirable, such as when the processing module 500disposed on the interface plate 660 comprises an incubator or otherheat-generating device, heat dissipating fins 664 may be provided on theinterface plate 660. To augment heat dissipation via the heatdissipating fins 664, the signal detection module 600 may include a fan670 disposed within a fan housing 672 mounted to the reformatter frame650. Fan 670 is constructed and arranged to generate air flow over theheat dissipating fins 664 to enhance the convective heat dissipationfrom the fins 664.

FIGS. 5 and 6 show front and rear, respectively, perspective views ofthe fiber reformatter frame 650 of the signal detection module 600 shownin FIGS. 2-4. The signal detector heads 200, the processing module 500,the fan 670, and the fan housing 672 are not shown in FIGS. 5 and 6. Thereformatter frame 650 includes sides 652, 654, a base 656 attached toone end of the sides 652, 654, and an interface plate 660 attached to anopposite end of the sides 652, 654. Signal coupling elements 662 areattached to each of the fiber-receiving openings formed in the interfaceplate 660. As explained above, coupling elements 662, which may compriseferrules, are constructed and arranged to couple a signal, e.g., anoptic signal, from the corresponding transmission fiber 180 to an objectto be interrogated, such as the contents of a receptacle, and/or couplean optical emission from the object into the transmission fiber 180.

The base 656 includes two openings 655, 657, each configured toaccommodate one of the signal detector heads 200. A plurality offiber-positioning holes 658 is provided around each of the openings 655,657. FIGS. 5 and 6 show only a portion of each of the transmissionfibers 180 extending from the interface plate 660. In the illustratedembodiment, the transmission fibers 180 are connected to the interfaceplate 660 in a rectangular configuration, and the fiber-positioningholes 658 formed in the base 656 are in a circular configuration so asto reformat the transmission fibers 180 from the rectangularconfiguration at the first ends thereof to a circular configuration atthe second ends thereof.

FIG. 7 is a perspective view of an alternative embodiment of areformatter frame 750. Reformatter frame 750 includes sides 752, 754 anda base 756 having an opening 755 formed therein with a plurality offiber-positioning holes 758 positioned around the opening 755 in agenerally circular configuration. An interface plate 760 is attached tothe sides 752, 754 of the frame 750 at an end thereof opposite the base756. Interface plate 760 includes a plurality of coupling elements 762,e.g., ferrules, and may include heat dissipating fins 764 disposed on aside of the interface plate 760 opposite the coupling elements 762. Eachcoupling element 762 corresponds to a fiber-receiving opening (notshown) formed through the interface plate 760. As can be seen in FIG. 7,the coupling elements 762 are arranged in a rectangular configuration ofsix rows of five coupling elements 762 each. The number of openings 758formed in the base 756 preferably corresponds to the number of couplingelements 762 formed in the interface plate 760. Thus, it can beappreciated that the reformatter frame 750 shown in FIG. 7 has half thecapacity of the reformatter frame 150 shown in FIG. 1, and that thereformatter frame 150 corresponds essentially to a doubling of thereformatter frame 750 with a second opening 755 and correspondingfiber-positioning holes 758 surrounding the opening and six additionalrows of five coupling elements 762 attached to the interface plate 760.However, one of skill in the art would appreciate that reformatter frame750 could be configured to have the same capacity, or more or lesscapacity to that of reformatter frame 150 shown in FIG. 1.

FIG. 8 shows an exemplary mapping of the spatial arrangement of fiberpositions in the interface plate 760 of the reformatter frame 750. Asshown in FIG. 8, the interface plate 760 includes six rows, or banks, offive fiber positions each, designated T1-T5, T6-T10, T11-T15, T16-T20,T21-T25, and T26-T30.

FIG. 9 shows a mapping of the spatial arrangement of fiber positions ofthe fiber-positioning holes 758 formed in the base 756 of thereformatter frame 750. In the illustrated embodiment, 35fiber-positioning holes 758 are formed in the base 756, and aredesignated F1, F2, F3, F4, . . . F35, starting at the lower (sixo'clock) position with respect to the opening 755.

FIG. 10 is a table showing an exemplary mapping of therectangularly-arranged interface positions T1-T30 in the interface plate760 to thirty of the circularly-arranged fiber-positioning holepositions F1-F35 in the base 756. This is exemplary only; other mappingsbetween the fiber positions in the interface plate 760 and the fiberpositions in the base 756 are contemplated. In this embodiment, thenumber of interface positions in the interface plate 760 is exceeded bythe number of fiber-positioning holes in the base 756 (e.g., 30 vs. 35).Fluorescent calibration targets can be placed in the additionalfiber-positioning holes in the base to test and/or calibrate the signaldetectors of the signal detector head 200.

In an alternative embodiment, the number of interface positions in theinterface plate 760 is equal to the number of fiber-positioning holes inthe base 756 (e.g., 30). It has been determined that theautofluorescence of the signal transmission fibers can also be used as afluorescent calibration target. For example, autofluorescence of thesignal transmission fibers can be used to determine the rotary positionsof the detector carrier 250 at which signal measurements should betaken. An exemplary process is as follows.

Starting at a known rotary position, e.g., as determined by a home flagassociated with the detector carrier 250, the detector carrier 250 canbe rotated, counting steps of the motor 352, until the autofluorescencesignal detected by each signal detector 300—each of which may beconfigured to detect a signal of a different wavelength—reaches a peak.Due to manufacturing and assembly tolerances, the number of motor stepsat which each signal detector detects a peaks signal may be somewhatdifferent. For example, in a system including five signal detectors 300,one signal detector 300 may peak at 130 steps from the home flagposition, another at 131 steps, another at 132 steps, another at 129steps, and another at 130 steps. The calibrated position at which ameasurement is taken may be determined as to be the closest whole numberof steps to the average of the five measurements, i.e., 130 steps (froman average of 130.4 steps) from the home position. If the tolerances inthe placement of the fiber positioning holes 758 are sufficiently small,so that the number of motor steps between fibers is known andrepeatable, no further calibration is necessary. Subsequent measurementscan be taken every known number of steps after the calibrated positionof the first measurement. If the tolerances are not sufficiently small,measurement positions for all fibers can be calibrated in a similarmanner—i.e., by stepping off the motor for each fiber position andtaking an average of the number of steps at which the signal detectorsdetect peak signals. It may be desirable to perform this calibrationprocedure at final assembly of the apparatus, at laboratory installationof the apparatus, after any service is performed on the apparatus, orbefore each time the apparatus is operated. FIG. 11 shows an alternativeembodiment of a thirty-fiber reformatter frame 850, including sides 852,854, a base 856 with an opening 855 and fiber-positioning openings 858surrounding opening 855, and an interface plate 860 having couplingelements 862 and heat dissipating fins 864 connected to an end of theframe 852 opposite the base 856. Fiber reformatter frame 850 iscomparable to the frame 750 shown in FIG. 7 and accommodates thirtytransmission fibers (not shown in FIG. 11) configured at the first endsthereof at the interface plate 860 in a rectangular configuration of sixrows of five fibers each and configured at the second ends thereof atthe base 856 in a circular configuration disposed within thefiber-positioning holes 858 surrounding the opening 855. The reformatterframe 850 shown in FIG. 11 differs from the reformatter frame 750 shownin FIG. 7 in that the base 856, the opening 855, and fiber-positioningopenings 858 are substantially centered with respect to the interfaceplate 860. In the reformatter frame 750 shown in FIG. 7, on the otherhand, the base 756, openings 755, and fiber-positioning openings 758 arelaterally offset with respect to the center of the interface plate 760.

Signal Detector Head

The signal detector head 200 is shown in FIG. 12. The signal detectorhead 200 may be attached to a reformatter frame (150, 650, 750, 850) andis constructed and arranged to index one or more signal detectors intooperative positions with respect to each transmission fiber disposed ina fiber-positioning hole of the base of the reformatter frame. Although,signal detector head 200 is configured to be coupled to any reformatterframe, including reformatter frames 150, 650, 750 and 850 describedherein, for simplicity of the description, the signal detector head 200will be described in the context of its implementation on reformatterframe 150 shown in FIG. 1.

In the embodiment shown in FIG. 12, the signal detector head 200includes a base plate 220 configured to be attached to the base 156 ofthe reformatter frame 150 and including a plurality of fiber tunnels 226arranged in a configuration corresponding to the spatial arrangement offiber-positioning holes 158 formed in the base 156 of the reformatterframe 150 so that each fiber tunnel 226 will align with a correspondingone of the fiber-positioning holes 158.

In general, the signal detector head is configured to move one or moresignal detectors to sequentially place each signal detector into anoperative position with respect to each transmission fiber 180 to detecta signal transmitted by the transmission fiber. The signal detector head200 further includes a detector carrier 250, which, in the illustratedembodiment, comprises a carousel that carries a plurality of signaldetectors 300 in a circular pattern. In the illustrated embodiment, thesignal detector head 200 includes six individual signal detectors 300,each mounted on a printed circuit board 210 and each configured toexcite and detect a different emission signal or an emission signalhaving different characteristics.

As will be described in further detail below, the detector carrier 250is configured so as to be rotatable with respect to the base plate 220.A detector drive system 350 constructed and arranged to effect poweredmovement, e.g., rotation, of the detector carrier 250 includes a drivemotor 350 supported on a motor mount portion 224 of the base plate 220.A drive belt 358 is disposed on an output shaft wheel 354 of the motor352 and around a pulley wheel 356 that is attached to or part of thedetector carrier 250. As can be appreciated, rotation of the outputshaft wheel 354 of the motor 352 causes a corresponding rotation of thepulley wheel 356 and the detector carrier 250 via the belt 358.

As would be further appreciated by persons of ordinary skill in the art,the configuration of the detector drive system 350 is exemplary, andother mechanisms and arrangements may be employed to effect poweredmovement of the detector carrier 250. For example, the output shaftwheel 354 may comprise an output gear that directly engages gear teethformed about the outer periphery of the pulley wheel 356, or the pulleywheel 356 could be coupled to the output shaft wheel 354 indirectly by agear train comprising one or more intermediate gears between the outputshaft wheel (gear) 354 and the pulley wheel 356. Alternatively, a drivemotor could be configured with its rotating output shaft attachedconcentrically to the detector carrier 250 and its axis of rotation sothat rotation of the output shaft by the motor causes a directcorresponding rotation of the detector carrier 250. Other arrangementsand configurations for effecting powered movement of the detectorcarrier 250 will be appreciated by persons of ordinary skill in the art.In particularly preferred embodiments, the detector carrier 250 anddetector drive system 350 are configured to provide for rotation of thedetector carrier 250 in a single direction.

Motor 352 is preferably a stepper motor and may include a rotaryencoder. The detector carrier 250 may include one or more positional orstatus feedback sensors. For example, the detector carrier 250 mayinclude a home flag 360 that is detected by an optical detector 362 forindicating a rotational “home” position of the carrier 250. Opticalsensor 360 may comprise a slotted optical sensor comprising an opticaltransmitter and receiver in which the path between the transmitter andreceiver is broken by the passage of the home flag 360. Persons ofordinary skill in the art will recognize, however, that other sensorsfor indicating a home position may be used. Such sensors may compriseproximity sensors, magnetic sensors, capacitive sensors, etc.

A rotary connector transmits data and/or power signals between therotating detector carrier 250 and the signal detectors 300 carriedthereon, and a non-rotating reference environment, such as a controllerand power source as described in more detail below. In the illustratedembodiment, the base 220 of the signal detector head 200 includescylindrical housing 222 projecting upwardly from a planar portion of thebase 220, and a slip ring connector 370 is positioned at an end of thecylindrical housing 222. The slip ring connector 370 includes a rotatingelement disposed inside the cylindrical housing 222 and a non-rotatingelement 372, attached or otherwise coupled to the non-rotatingcylindrical housing 222 by an intermediate ring 376, to which areattached data/power cables 374. The slip ring connector 370 transmitsdata and/or power signals between the rotating detector carrier 250 andthe signal detectors 300 carried thereon, and a non-rotating referenceenvironment, such as a controller and power source as described in moredetail below.

Further details of the signal detector head 200 are shown in FIG. 13,which is a transverse cross-sectional view of the detector head 200along the line XIII-XIII in FIG. 12. Each signal detector 300 includes adetector housing 302 within which are formed an excitation channel 304and an emission channel 306, which, in the illustrated embodiment, aregenerally parallel to one another. An excitation source 308, such as anLED, is mounted on the printed circuit board 210 at the base of theexcitation channel 304. An emission detector 314, such as a photodiode,is coupled to the printed circuit board 210 and is disposed within theemission channel 306.

The detector carrier 350 further includes, positioned adjacent thesignal detector housing 302, a filter plate 264 having a central opening276 formed therein and defining an annulus. Within the annulus, anemission filter opening 282 and an excitation filter opening 280 areformed in alignment with the emission channel 306 and the excitationchannel 304, respectively, of each signal detector housing 302. Anexcitation lens 310 and an excitation filter 312 are disposed in theexcitation opening 280. Although a single excitation lens 310 and asingle excitation filter 312 are shown in FIG. 13, the signal detector300 may include multiple excitation filters and/or multiple excitationlenses. Similarly, an emission filter 316 and an emission lens 318 aredisposed in the emission opening 282. Although a single emission filter316 and a single emission lens 318 are shown in FIG. 13, the signaldetector 300 may include multiple emission lenses and/or multipleemission filters.

The detector carrier 250 further includes, adjacent the filter plate264, a mirror plate 260 having a central opening 262 and defining anannulus. The annulus of the mirror plate 260 has formed therein openingsaligned with the emission opening 282 and the excitation opening 280formed in the filter plate 264 for each signal detector 300. A mirror320 is disposed in the mirror plate 260 in general alignment with theexcitation channel 304, and a dichroic filter 322 is disposed in themirror plate 260 in general alignment with the emission channel 306.Mirror 320 is oriented at an angle (e.g. 45°) with respect to theexcitation channel 304 so as to be configured to redirect a light beam.

The detector carrier 250 further includes an objective lens plate 252having a central opening 256 formed therein and defining an annulus. Alens opening 254 is formed through the annulus of the objective lensplate 252 in general alignment with the emission channel 306 of eachsignal detector 300. An objective lens 324 is disposed within the lensopening 254.

The base plate 220 is disposed adjacent the objective lens plate 252 andincludes fiber tunnels 226 formed about the perimeter thereof. Althoughbase plate 220 and objective lens plate 252 are depicted as abuttingone-another in FIG. 13, it is contemplated that there can be adesignated distance, forming an air gap, between the base plate 220 andthe objective lens plate 252. Also, objective lens plate 252 and mirrorplate 260 are depicted as abutting one-another in FIG. 13, it iscontemplated that there can be a designated distance, forming an airgap, between the objective lens plate 252 and the mirror plate 260.

The detector carrier 250, comprising the objective lens plate 252, themirror plate 260, and the filter plate 264, as well as the signaldetectors 300 carried thereon, are rotatable with respect to the baseplate 220 so that each objective lens 324 associated with each of thesignal detectors 300 can be selectively placed into operative alignmentwith one of the fiber tunnels 226 disposed in the base plate 220. Thus,in the illustrated embodiment having six signal detectors 300, at anygiven time, six of the fiber tunnels 226 are in operative, opticalalignment with one of the objective lenses 324 and its correspondingsignal detector 300.

Operation of the signal detector 300 in an exemplary embodiment isillustrated schematically in FIG. 14. The detector 300 shown is afluorometer that is constructed and arranged to generate an excitationsignal of a particular, predetermined wavelength that is directed at thecontents of a receptacle to determine if a probe or marker having acorresponding emission signal of a known wavelength is present. When thesignal detector head 200 includes multiple fluorometers—e.g., six—eachfluorometer is configured to excite and detect an emission signal havinga different wavelength to detect a different label associated with adifferent probe hybridized to a different target analyte. When a morefrequent interrogation of a sample is desired for a particular emissionsignal, it may be desirable to incorporate two or more fluorometersconfigured to excite and detect a single emission signal on the signaldetector head 200.

An excitation signal is emitted by the excitation source 308. Excitationsource, as noted above, may be an LED and may generate light at apredetermined wavelength, e.g. red, green, or blue light. Light from thesource 308 passes through and is focused by an excitation lens 310 andthen passes through the excitation filter 312. As noted, FIG. 15 is aschematic representation of the signal detector 300, and the focusingfunctionality provided by the excitation lens 310 may be effected by oneor more separate lenses disposed before and/or after the filter element312. Similarly, the filter functionality provided by the filter element312 may be effected by one or more individual filters disposed beforeand/or after the one or more lenses that provide the focusingfunctionality. Filter element 312 may comprise a low band pass filterand a high band pass filter so as to transmit a narrow wavelength bandof light therethrough. Light passing through the excitation lens 310 andexcitation filter element 312 is reflected laterally by the mirror 320toward the dichroic 322. The dichroic 322 is constructed and arranged toreflect substantially all of the light that is within the desiredexcitation wavelength range toward the objective lens 324. From theobjective lens 324, light passes into a transmission fiber 180 andtoward the receptacle at the opposite end thereof. The excitation signalis transmitted by the transmission fiber 180 to a receptacle so as toexpose the contents of the receptacle to the excitation signal.

A label that is present in the receptacle and is responsive to theexcitation signal will emit an emission signal. At least a portion ofany emission from the contents of the receptacle enters the transmissionfiber 180 and passes back through the objective lens 324, from which theemission light is focused toward the dichroic 322. Dichroic 322 isconfigured to transmit light of a particular target emission wavelengthrange toward the emission filter 316 and the emission lens 318. Again,the filtering functionality provided by the emission filter 316 may beeffected by one or more filter elements and may comprise a high bandpass and low band pass filter that together transmit a specified rangeof emission wavelength that encompasses a target emission wavelength.The emission light is focused by the emission lens 318, which maycomprise one or more lenses disposed before and/or after the filterelements represented in FIG. 14 by emission filter 316. The emissionlens 318 thereafter focuses the emission light of the target wavelengthat the detector 314. In one embodiment, the detector 314, which maycomprise a photodiode, will generate a voltage signal corresponding tothe intensity of the emission light at the prescribed target wavelengththat impinges the detector.

Returning again to FIG. 13, a flanged tube 266 extends through thecentral opening 256 of the objective lens plate 252 and through thecylindrical housing 222 of the base plate 220. The flanged tube 266includes a cylindrical tube 268 extending through the central opening256 and the cylindrical housing 222 and a radial flange 270 disposedwithin the central opening 262 of the mirror plate 260 and secured bysuitable fasteners, such as screws or bolts, to the objective lens plate252. Longitudinally-spaced bearing races 272, 274 are disposed betweenthe interior of the cylindrical housing 222 and the exterior of thecylindrical tube 268 of the flanged tube 266. Thus, as can beappreciated, the flanged tube 266 will rotate, with the detector carrier250, with respect to the base plate 220 and the cylindrical housing 222.

Further details of an exemplary representation of the slip ring 370 arealso shown in FIG. 13. The slip ring connector 370 is disposed at theend of the cylindrical tube 268 opposite the radial flange 270. As notedabove, the cylindrical tube 268 rotates with the detector carrier 250,while the cylindrical housing 222 remains stationary with the base plate220. The slip ring connector 370, which may comprise slip rings andbrushes as are known, includes stationary components attached orotherwise coupled to the cylindrical housing 222 and rotating componentsattached or otherwise coupled to the rotating cylindrical tube 268. Ingeneral, components 372, 376 represent non-rotating portion(s) of theslip ring 370 in which fixed contact components, such as the brush(es),are located, component 378 located inside tube 268 represents rotatingportion(s) of the slip ring 370 that rotate with the tube 268 and inwhich rotating contact elements, such as the ring(s) are located, andcable 379 represents a power and/or data conductor(s) connectingcomponent 378 with the printed circuit board 210 and which rotates withthe printed circuit board 210 and the signal detector carrier 250.

As the detector carrier 250 rotates, each of the signal detectors 300 issequentially placed in an operative position with respect to a secondend of a different transmission fiber 180 to interrogate (i.e., measurea signal from) an emission signal source located at a first end of thetransmission fiber 180. The detector carrier 750 pauses momentarily ateach transmission fiber 180 to permit the signal detector 300 to detectan emission signal transmitted through the transmission fiber 180. Wherethe signal detector 300 is a fluorometer, the detector carrier pausesmomentarily to permit the signal detector to generate an excitationsignal of a specified wavelength that is transmitted by the transmissionfiber 180 to the emission signal source (receptacle) and to detectfluorescence of a specified wavelength excited by the excitation signalthat is emitted by the contents of the receptacle and transmitted by thetransmission fiber 180 to the fluorometer. Thus, in an embodiment, eachtransmission fiber 180 can be employed to transmit both an excitationsignal and the corresponding emission signal, ad each signal detectorcan be used to scan multiple transmission fibers and associated emissionsignal sources.

The emission signal source associated with each transmission fiber 180is interrogated once by each signal detector 300 for every revolution ofthe detector carrier 250. Where the signal detector head 200 includesmultiple signal detectors 250 configured to detect different signals,each emission signal source is interrogated once for each differentsignal for every revolution of the detector carrier. Thus, in the caseof a nucleic acid diagnostic assay, which may include PCR amplification,the contents of each receptacle is interrogated for each target analytecorresponding to the different probes employed (as indicated bydifferent colored labels) once for each revolution of the detectorcarrier 250.

In one embodiment, in which base plate 220 of the signal detector head200 includes thirty (30) fiber tunnels for thirty (30) transmissionfibers 180, the signal detector carrier rotates one revolution everyfour (4) seconds, stopping at least ten (10) milliseconds at each fibertunnel to measure an emission signal transmitted by the associatedtransmission fiber. Again, if the signal detector head include multiplesignal detectors (e.g., six (6) fluorometers), the signal detector headwill measure an emission for each of the six different wavelengths ofinterest once every four (4) seconds. Accordingly, time vs. emissionsignal intensity data can be generated for each receptacle for eachwavelength.

When performing PCR, it is not necessary to synchronize the signal dataacquisition with the thermal cycles of the PCR process. That is, it isnot necessary that the emission signal of each receptacle be measured atthe same temperature point (e.g., 95° C.) in the PCR cycle. By recordingdata every four seconds during the entire PCR process, a sufficientnumber of data points will be collected at each temperature of the PCRthermal cycle. The signal emission data is synchronized with specifictemperatures by recording a time stamp for each emission signalmeasurement and a time stamp for each temperature of the thermal cyclingrange. Thus, for example, to identify all signal measurements occurringat a temperature of 95° C., the time stamps of the signal measurementsare compared to the temperature time stamps corresponding to atemperature of 95° C.

The time duration of a thermal cycle is variable, depending on the assaybeing performed. The minimum time interval is dictated by how fast thethermocycler can ramp temperatures up and down. For a cycler that canramp the vial filled with fluid from 55° C. to 95° C. in about 15seconds, an exemplary cycle would be anneal at 55° C. for 25 seconds, a15 second from 55° C. to 95° C., denature at 95° C. for 5 seconds, and15 second ramp back down from 95° C. to 55°, and then begin anothercycle with a 25 second anneal, Thus, this exemplary anneal-denaturecycle would be a 60 second cycle.

The control and data acquisition system of the signal detector head 200is shown schematically in FIG. 15. As shown in FIG. 15, the detectorcarrier 250 carries one or more signal detectors 300, each of which may,in one embodiment, include an excitation source 308, an excitation lens310, a mirror 320, a dichroic 322, an objective lens 324, an emissionlens 318, and an emission detector 314 as described above. Eachreceptacle 504 carried in, e.g., a processing module 500 (see FIGS.2-4), is coupled to a transmission fiber 180 that terminates in the baseplate 220 of the signal detector head 200. Motor 352 is mechanicallycoupled to the detector carrier 250 by a motor coupler 380 to effectpowered movement (e.g., rotation) of the detector carrier 250. Acontroller 810 may be coupled to a controllable power source 800 and tothe motor 352 for providing motor control signals and receiving motorposition feedback signals, e.g., from a rotary encoder. Controller 810may also be coupled to other feedback sensors, such as the home sensor360, for detecting a rotational position of the detector carrier 250.Controller 810 also provides controlled power signals, via the slip ringconnector 370, to the excitation sources 308 rotatably carried on thedetector carrier 250 and coupled to the printed circuit board 210. Thefunctionality of controller 810 may be provided by one controller ormultiple controllers in functional communication with each other.Moreover, one or more controllers, or one or more component(s) thereof,may be carried on the rotating portion of the detector head 200, such ason the printed circuit board 210. Voltage signals from the emissiondetectors 314, coupled to the printed circuit board 210, and other datamay be carried from the detector carrier 250, via the slip ringconnector 370, to a processor 820 for storing and/or analyzing the data.Alternatively, processor 820, or one or more component(s) thereof, maybe carried on the rotating portion of the detector head 200, such as onthe printed circuit board 210.

An exemplary control configuration of the signal detector head 200 isrepresented by reference number 900 in FIG. 16. An optics controller 902may be provided for each detector carrier, or rotor, and coupled to theprinted circuit board 210 to which the excitation sources (LED) 308 andemission detectors (PD (photodiode)) 314 are attached. Each opticscontroller 902 may include a microcontroller 912, e.g., a PIC18F-seriesmicrocontroller available from Microchip Technology Inc., an analog todigital converter 906, and an integrated amplifier 908 (e.g., one foreach emission detector (PD) 314). A constant current driver 910 (e.g.one for each excitation source 308) is controlled by the microcontroller928 and generates control signals (e.g., controlled power) to theexcitation source 308. Controller 902 receives power at 916 (e.g., 24 V)from the slip ring connector 370 and includes a serial data link RS-485914 for commutations between the controller 902 and the slip ringconnector 370.

An exemplary control configuration 900 may include a motion controller920 for each detector drive 350 (see FIG. 12). At 932, motion controller920 receives power, e.g., 24 VDC, 40 watts from controllable powersource 800 (see FIG. 15), that is transmitted to the optics controller902 via the slip ring 370. Motion controller 920 may communicate with anexternal controller via a serial data link 930. In one embodiment,controller 920 communicates with a controller of the thermocycler tosynchronize operation of the signal detector head 200 with operation ofthe thermocycler. Controller 920 may include a serial data link RS-485926 for communications between the controller 920 and the slip ring 370.Controller 920 may further include a microcontroller 928, e.g., aPIC18F-series microcontroller available from Microchip Technology Inc.and a PMD chip set 924, which is a motor controller to control thestepper motor. A stepper motor driver 936 is controlled by themicrocontroller 928 and generates motor control signals for the motor352 of the optics rotor (i.e., detector drive). A slotted optical sensorinput 922 receives signals from the home flag sensor 362 andcommunicates such signals to the microcontroller 928.

An alternative embodiment of a signal detector head embodying aspects ofthe present disclosure is indicated by reference number 420 in FIGS. 22and 23. Signal detector head 420 includes a filter wheel 422 and acamera 450 oriented in a radial focal direction with respect to thefilter wheel 422. In general, signal detector head 420 employs thecamera 450 to image a plurality of bundled fibers to detect a signaltransmitted by each fiber. The filter wheel 422 can be indexed toselectively couple each of one or more excitation sources and emissionfilters with the fiber bundle and the camera 450 to direct an excitationsignal of a specified characteristic, e.g., wavelength, to the fibers ofthe fiber bundle and to direct emission signals of a specifiedcharacteristic, e.g., wavelength, from the fibers of the fiber bundle tothe camera 450.

More particularly, signal detector head 420 includes a filter wheel 422that comprises a body 424. Body 424 may be a body or assembly ofrevolution configured to be rotatable about a central axis. A motor 460is coupled to the filter wheel 422 by a transmission element 462 toeffect powered rotation of the filter wheel 422. Transmission element462 may comprise any suitable transmission means for transmitting therotation of the motor 460 to the filter wheel 422. Exemplarytransmissions include interengaged gears, belts and pulleys, and anoutput shaft of the motor 460 directly attached to the body 424, etc.Motor 460 may be a stepper motor to provide precise motion control andmay further include a rotary encoder. The filter wheel 422 may furtherinclude a home flag for indicating one or more specified rotationalpositions of the filter wheel 422. Suitable home flags include slottedoptical sensors, magnetic sensors, capacitive sensors, etc. A fiberbundle 452 includes a plurality of fibers fixed at the first endsthereof with respect to the filter wheel 422, e.g., to a fixed plate 442located adjacent to the filter wheel 424, by a fiber mounting block 456.The second ends of the respective fibers are coupled to each of aplurality of signal sources positioned in a first specified arrangement,and may include receptacles (such as receptacles 504) positioned in arectangular arrangement.

The filter wheel 422 includes one or more optics channels 425 and ismovable so as to selectively index each optics channel 425 into anoperative, optical communication with the fiber bundle 452 and thecamera 450. Each optics channel 425 includes an excitation channel 426formed in an axial direction within the body 424 of the index wheel 422for transmitting an excitation signal to the fiber bundle 452 and anemission channel 436 extending radially from the excitation channel 426to a radial opening on the outer periphery of the filter wheel 422.

An excitation source 428, e.g., a bright light LED, is disposed withinthe excitation channel 426. The excitations sources 428 of all theemission channels 436 may be connected to a printed circuit board 448.One or more lenses 430 and one or more excitation filters 432 arepositioned within the excitation channel 426 to condition light emittedby the source 428. Each optics channel 425 may be configured to generateand transmit an excitation signal of a specified wavelength. In such anembodiment, filter(s) 432 are configured to transmit light at thedesired wavelength.

Each channel 425 includes a dichroic 434 configured to transmit thatportion of the excitation signal that is at or near the prescribedexcitation wavelength.

When the optics channel 425 is in optical communication with the fiberbundle 452—such as by rotating he filter wheel 424 until the opticschannel 425 is aligned with a fiber tunnel 444 within, or adjacent to,which the fiber bundle 452 is secured—an objective lens 446 transmitsthe excitation signal from the excitation channel 426 into each fiber ofthe fiber bundle 452. Emissions from the emissions sources at theopposite ends of the fibers are transmitted by each fiber of the fiberbundle 452 back through the objective lens 446 and into the opticchannel 425. Dichroic 434 may be configured to reflect light of aspecified emission wavelength. Thus, that portion of the emission lighttransmitted by the fiber bundle 452 into the optics channel 425 that isat the specified emission wavelength is reflected by the dichroic 434into the emission channel 436.

An emission filter 438 is disposed within the emission channel 436 andis configured to transmit light having the desired emission wavelength.The emission channel 436 terminates at a radial opening formed about theouter periphery of the body 424. In an embodiment, the optics channel425 is oriented with respect to the camera 450 such that an opticchannel 425 that is in optical communication with the fiber bundle 452is also in optical communication with the camera 450.

When the optics channel 425 is an operative position with respect to thecamera 450, the radial opening of the emission channel 436 is alignedwith image relay optics 440 that transmit emission light from theemission channel 436 into the camera 450. Camera 450 then images theemission signals transmitted by all fibers in the fiber bundle 452 atonce. To determine the signal transmitted by each fiber—and thus thesignal emitted by the signal emission source associated with thefiber—the pixels of the camera's pixel matrix are mapped to the fiberlocations within the fiber bundle to identify the one or more pixels ofthe pixel array that correspond to each fiber. By interrogating thesignal imaged at each pixel or group of pixels associated with a fiber,the signal (e.g. the color (wavelength) and/or intensity) of the missionsignal transmitted by that fiber can be determined.

Suitable cameras include CMOS camera such as the IDS UI-5490HE camera orCCD camera such as the Lumenera LW11059 or the Allied GE4900.Preferably, the camera has at least 10 megapixels and has a high framerate.

In an embodiment, the filter wheel 422 includes multiple (e.g., 3 to 6)optics channels 425, each configured to excite and detect an emission ofa different wavelength or other specific, distinguishing characteristic.Thus by rotating the filter wheel to index each optics channel 425 withrespect to the fiber bundle 452 and camera 450, signals of eachdistinguishing characteristic can be measure from all fibers andassociated signal emission sources.

It will be appreciated that the signal detector head may include one ormore additional cameras positioned and be coupled to one or moreadditional fiber bundles to permit simultaneous imaging of the multiplefiber bundles.

Hardware and Software

Aspects of the disclosure are implemented via control and computinghardware components, user-created software, data input components, anddata output components. Hardware components include computing andcontrol modules (e.g., system controller(s)), such as microprocessorsand computers, configured to effect computational and/or control stepsby receiving one or more input values, executing one or more algorithmsstored on non-transitory machine-readable media (e.g., software) thatprovide instruction for manipulating or otherwise acting on the inputvalues, and output one or more output values. Such outputs may bedisplayed or otherwise indicated to a user for providing information tothe user, for example information as to the status of the instrument ora process being performed thereby, or such outputs may comprise inputsto other processes and/or control algorithms. Data input componentscomprise elements by which data is input for use by the control andcomputing hardware components. Such data inputs may comprise positionssensors, motor encoders, as well as manual input elements, such askeyboards, touch screens, microphones, switches, manually-operatedscanners, etc. Data output components may comprise hard drives or otherstorage media, monitors, printers, indicator lights, or audible signalelements (e.g., buzzer, horn, bell, etc.).

Software comprises instructions stored on non-transitorycomputer-readable media which, when executed by the control andcomputing hardware, cause the control and computing hardware to performone or more automated or semi-automated processes.

While the present disclosure has been described and shown inconsiderable detail with reference to certain illustrative embodiments,including various combinations and sub-combinations of features, thoseskilled in the art will readily appreciate other embodiments andvariations and modifications thereof as encompassed within the scope ofthe present invention. Moreover, the descriptions of such embodiments,combinations, and sub-combinations is not intended to convey that thedisclosures require features or combinations of features other thanthose expressly recited in the claims. Accordingly, the presentinvention is deemed to include all modifications and variationsencompassed within the spirit and scope of the following appendedclaims.

The invention claimed is:
 1. A method of measuring a time-varying signalemission, comprising: (a) subjecting the contents of a receptacle to athermal cycling process; (b) during step (a), measuring a signalemission from the contents of the receptacle at regular first timeintervals and recording the measured signal emission and a first timestamp at each time interval; (c) during step (a), determining atemperature of the thermal cycling process at regular second timeintervals and recording the determined temperature and a second timestamp at each time interval, wherein the regular first time intervalsand the first time stamps are different from the regular second timeintervals and second time stamps, respectively; and (d) synchronizingthe measured signal emissions with a specific temperature of the thermalcycling process by comparing the first time stamps of the measuredsignal emissions with the second time stamps of the determinedtemperatures that correspond with the specific temperature.
 2. Themethod of claim 1, wherein thermal cycling process is a PCR process. 3.The method of claim 2, wherein the thermal cycling process includes anannealing temperature and a denaturation temperature.
 4. The method ofclaim 3, wherein the specific temperature is the denaturationtemperature.
 5. The method of claim 4, wherein the specific temperatureis 95° C.
 6. The method of claim 2, wherein each cycle of the thermalcycling reaction is a 60 second cycle.
 7. The method of claim 1, whereinthe signal emission is a fluorescent emission.
 8. The method of claim 1,wherein the time intervals of step (b) are four seconds.
 9. The methodof claim 1, wherein step (d) comprises identifying all measured signalemissions at the specific temperature.